The prophenoloxidase-activating cascade is a key component of arthropod immunity. Drosophila prophenoloxidase is stored in crystal cells, a specialized class of blood cells from which it is released through cell rupture. Within minutes after bleeding, prophenoloxidase is activated leading to visible melanization of the clot matrix. Using crystal cell rupture and melanization as readouts to screen mutants in signal transduction pathways, we show that prophenoloxidase release requires Jun N-terminal kinase, small Rho GTPases and Eiger, the Drosophila homolog of tumor necrosis factor. We also provide evidence that in addition to microbial products, endogenous signals from dying hemocytes contribute to triggering and/or assembly of the prophenoloxidase-activating cascade, and that this process can be inhibited in vitro and in vivo using the viral apoptotic inhibitor p35. Our results provide a more comprehensive view of immune signal transduction pathways, with implications for immune reactions where cell death is used as a terminal mode of cell activation.

Clotting is critical in limiting loss of hemolymph and initiating wound healing in insects as well as in vertebrates. Clotting is also an important immune defense, quickly forming a secondary barrier to infection, thereby immobilizing, and possibly killing bacteria directly. Here, we describe methods to assess clotting and to extract the clot from Drosophila larval hemolymph by using aggregation of paramagnetic beads. The validity of the assay was demonstrated by characterization of mutants. We show that clotting occurs in the absence of phenoloxidase and that the Drosophila clot binds bacteria. We also describe a pullout assay to purify the clot as a whole, free from entrapped hemocytes and cellular debris. Proteins subsequently identified by mass spectrometry include both predicted and novel clot proteins. Immune induction has been shown for three of the latter, namely Tiggrin and two unknown proteins (GC15825 and CG15293) that we now propose function in hemolymph clotting. The most abundant clot protein is Hemolectin, and we confirm that hemolectin mutant larvae show clotting defects.

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The enzyme phenoloxidase (PO) is one of the first immune molecules that was identified in invertebrates. Recently, the immune function of PO has been challenged. We tested how PO is activated following injury in 2 insects, i.e. the fruit fly Drosophila melanogaster and the wax moth Galleria mellonella. Rapid PO activation in Drosophila was limited to discrete areas of the hemolymph clot which forms after injury. Surprisingly, unlike systemic PO activation during bacterial sepsis, clot melanization was not sensitive to microbial elicitors in our assay. Instead, Drosophila clot melanization was activated by endogenous signals such as apoptotic cells and was superinduced by phosphatidylserine, a negatively charged phospholipid normally found on the inner surface of the plasma membrane and exposed during apoptosis. In contrast, melanization in G. mellonella hemolymph was stronger and more uniform and was sensitive to peptidoglycan. This shows that both exogenous and endogenous signals can trigger the same immune mechanism in species- and context-dependent ways. Our findings have implications for the evolutionary dynamics of immune mechanisms and are in agreement with recent comparisons of insect immune transcriptomes.